Study of antigen (Ag)-specific T cell responses poses formidable technical challenges [Kern, Trends Immunol. 26:477, 2005]. This is mainly due to the fact that Ag-specific fractions are commonly represented at very low frequencies in peripheral blood, a feature which makes their detection troublesome [Mallone, Clin. Immunol. 110:232, 2004]. This detection is even more problematic when CD4+ T cells are considered, as these fractions are frequently present at even lower frequencies than their CD8+ counterparts [Homann, Nat. Med. 7:913, 2001; Seder, Nat. Immunol. 4:835, 2003].
Several detection strategies are currently available which allow to detect such Ag-specific T cells (CD4+ and CD8+) using a variety of structural or functional readouts [Kern, Trends Immunol. 26:477, 2005]. However, one drawback shared by all techniques is that Ag-specific CD4+ T cells can rarely be detected directly ex-vivo. Most commonly, these cells need to be preliminarily expanded through 5-14 d in vitro culture steps to reach the detection threshold [Mallone, Clin. Immunol. 110:232, 2004]. A number of approaches can be used for this in vitro expansion. As peripheral blood mononuclear cells (PBMCs) contain suitable numbers of CD4+ T cells as well as antigen-presenting cells (APCs; monocytes, B cells, and minute fractions of circulating dendritic cells) (DC), they can be pulsed with the peptide epitopes or protein Ags of interest and expanded with or without the addition of co-stimulatory cytokines such as interleukin (IL)-2 and IL-7.
Alternatively, monocytes can first be isolated and differentiated into immature DC with granulocyte/macrophage colony-stimulating factor (GM-CSF) and IL-4 for 5-7 days, to be subsequently matured with different proinflammatory stimuli for an additional 24-48 h [Zhou et al., Proc. Natl. Acad. Sci. USA 93:2588, 1996]. This strategy exploits the higher stimulatory potency of DCs to achieve a larger CD4+ T cell expansion. While attractive, it requires however larger starting blood volumes, as monocytes represent only ˜5-15% of PBMCs, and autologous monocytes should ideally be used to avoid selection of allo-specific CD4+ T cells. T cells need therefore to be kept in culture or frozen down while monocyte-derived DC are being generated. Besides higher PBMC needs, this procedure is also longer than those relying exclusively on blood natural APCs.
Moreover, use of peptide epitopes for T-cell stimulation requires preliminary identification of those epitopes targeted by the immune response. This identification procedure is very labor-intensive, and specific for one HLA Class I or Class II allele. Thus, different epitopes have to be identified for different HLA alleles, depending on the human subjects that one wishes to study.
Of further note, it is frequently of interest not only to detect CD4+ T cells, but also to isolate and expand them for further functional profiling.
Therefore, there is still an unmet need in the art for providing a sensitive, versatile and easy-to-use method for measuring T cell responses and for isolating T cell clones, in particular CD4+ T cells.